- Comparative analysis of Cd-responsive maize and rice transcriptomes highlights Cd co-modulated orthologs. - Results: To unveil conserved Cd-responsive genes in cereal plants, the obtained 5166 maize DEGs were compared with 2567 Cd-regulated orthologs in rice roots, and this comparison generated 880 universal Cd-responsive orthologs groups composed of 1074 maize DEGs and 981 rice counterparts. - More importantly, most of the orthologous DEGs showed coordinated expression pattern between Cd-treated maize and rice, and these include one large orthologs group of pleiotropic drug resistance (PDR)-type ABC transporters, two clusters of amino acid transporters, and 3 blocks of multidrug and toxic compound extrusion (MATE) efflux family transporters, and 3 clusters of heavy metal-associated domain (HMAD) isoprenylated plant proteins (HIPPs), as well as all 4 groups of zinc/iron regulated transporter protein (ZIPs).. - Out of the 1074 maize DEGs, approximately 30 maize Cd-responsive genes such as ZmHIPP27, stress-responsive NAC transcription factor (ZmSNAC1) and 9-cis-epoxycarotenoid dioxygenase (NCED, vp14) were also common stress-responsive genes reported to be uniformly regulated by multiple abiotic stresses. - Moreover, the aforementioned three promising Cd- upregulated genes with rice counterparts were identified to be novel Cd-responsive genes in maize.. - Conclusions: These novel findings revealed the conserved function of Cd-responsive orthologs and paralogs, which would be valuable for elucidating the genetic basis of the plant response to Cd stress and unraveling Cd tolerance genes.. - 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0. - As for the other model cereal maize, a few RNAseq-based transcriptomic studies focusing on Cd-responsive genes have been conducted [18, 19]. - However, to the best of our knowledge, there is no report on the identification of universal cereal Cd-responsive genes, in other words, Cd co-modulated orthologs between maize and rice. - In the current study, we firstly implemented the Tophat-Cufflinks pipeline to identify early Cd-responsive DEGs in maize and rice seedlings roots. - After that, the Cd-responsive maize orthologs with syner- gistically Cd-regulated rice counterparts were queried against multiple stress common-responsive gene sets, and. - 30 DEGs in the intersection are of particular interest, in- cluding those encoding HMAD isoprenylated protein ZmHIPP27, transcription factor ZmSNAC1, and key en- zyme for ABA synthesis. - Further Cd-tolerance assay in yeast and tobacco leaves indicated that ZmGAD1, which had Cd co-modulated rice ortholog, exhibited Cd toler- ance in the host cells. - These results could lead to a com- prehensive understanding of the genetic basis of the plant response to Cd stress and open prospective for excavating. - Over 80% of the clean reads had scores at the Q30 level (Additional file 1: Table S1).. - The primers used in the qRT-PCR experiments are listed in Additional file 2: Table S2.. - Pre-cultured cells were diluted to an OD 600 of 1.0, and 10-μL aliquots were spotted onto SD-Ura agar medium with or without 40 μM CdCl 2 in the presence of 2% galactose. - RFP-H2A, localized in the nu- cleus, was used to mark the nuclei [36]. - After 3 days of agro-infiltration, the needle hole in the leaves expressing ZmGAD- GFPs were re-infiltrated with 500 μM Cd(NO 3 ) 2. - Functional characterization of early cd-responsive differentially expressed genes (DEGs) in maize roots To investigate transcripts that were specifically regulated in short-term Cd stressed maize roots, RNAseq data from replicated samples were processed through TopHat-Cuf- flinks pipeline to perform pair-wise comparisons between 1 h Cd-treated (Cd1h) and untreated (ck1h) maize seed- lings (Additional file 1: Table S1).. - 1.5 and q_value≤0.05), a total of 5166 genes were identified as being early Cd-responsive differentially expressed genes (DEGs), of which 3715 were Cd-induced and 1451 were Cd-repressed in maize seedlings roots (Additional file 3: Table S3). - However, only 239 DEGs were in the intersection of these 5166 and the avail- able 768 Cd-responsive genes in B73 or Mo17 across three time points reported previously [19] (Additional. - To gain insights into the functionality of the 5166 DEGs that are likely to be associated with the Cd re- sponse, all of these Cd-responsive transcripts were func- tionally grouped and visualized in the candidate pathway networks with MapMan software.. - Among the DEGs within the ‘TF’ group, 9 members of C2C2(Zn) DOF zinc finger family, 11 HSFs (including those in the subcategory “heat” of ‘Stress’ group), 11 members of Triple-Helix TFs family, and the majority of EREBP and NAC as well as WRKY family TFs were up- regulated in response to Cd (Fig. - Of the transcripts mapped to ‘Hormones’ category, 17 genes (including 7 lipoxygenase LOX, 5 allene oxidase synthase AOS, one allene oxidase cyclase and four 12-oxophytodienoate reductases OPR) for jasmonate synthesis, and 6 genes for brassinosteroid metabolism and signaling were also upregulated by Cd treatment.. - Conserved cd-responsive orthologous genes in maize and rice roots. - Therefore, the global comparison of the DEGs identified in the short-term Cd treated maize and rice roots (NCBI-SRA SRP053169, Additional file 5: Table S5) was performed with the aid of plant model organism orthologs annotation [37, 38].. - This comparison output 1074 Cd-responsive maize orthologs of 981 rice genes, which can be categorized. - 1 Global view of 5166 DEGs involved in diverse metabolic pathways in maize roots under Cd stress. - Moreover, 80 of them are in the list of 768 Cd-responsive genes in B73 or Mo17 roots under vari- ous Cd pressures [19]. - Conversely, 994 maize DEGs with rice counterparts were not stated as Cd-responsive genes previously (Additional file 6: Table S6). - Among the 80 universal Cd-responsive DEGs, 4 GLP members in group MCL23, 2 patatin-like members of MCL12549, and two 12-oxo-phytodienoic acid reductases (ZM2G000236 and ZM2G087192 designated as ZmOPR2 and ZmOPR5, re- spectively) in cluster MCL166 were also significantly up- regulated in both maize genotypes (Table 1) [19].. - To further explore the common regulatory mechanisms in maize and rice under Cd stress, these Cd-responsive orthologs were investigated their involvement in various metabolic pathways. - According to the global function view of these Cd-responsive orthologs in MapMan, the major- ity of them were upregulated in response to Cd, whether in maize or in rice roots (Additional file 7: Figure S1).. - DEGs underlined are also Cd-responsive reported previously [19]. - Table 2 Cd-regulated DEGs of orthologous transporters in maize and rice roots. - Those genes underlined are also Cd-responsive DEGs in previous report [19]. - Cd-responsive patatin-like storage proteins (ZM2G114036 and ZM2G414047) reported previously [19] are reverse tandem paralogs on chromosome 1 (Table 1).. - However, it is noteworthy that 25 of the 28 Cd-responsive maize transporters with co-modulated rice orthologs were not demonstrated as Cd-responsive genes previously (Table 2).. - Common stress-responsive genes in maize. - To ascertain whether these conserved Cd-responsive maize genes are involved in diverse stress response, they are compared with those identified in previous RNAseq analysis of maize seedlings subjected to drought, salinity and cold [39, 40]. - Remarkably, out of 1074 Cd-responsive maize DEGs with rice counterparts, about 30 genes are also in the list of DEGs response to these abiotic stresses, and they can be grouped into 5 categories. - Among the 30 common stress-responsive genes, only ZM2G172230 encoding CaseinoLytic Protease (ClpD) and ZM2G328785 coding receptor protein kinase (ZmPK1), both in the category ‘Posttranslational modification. - ZmGADs confer cd tolerance in cd-sensitive yeast mutant To unveil novel Cd-tolerant genes from the Cd-responsive orthologs in maize, the key enzyme re- sponsible for γ-aminobutyric acid (GABA) synthesis named glutamate decarboxylase (GAD) was selected, since Cd-regulated GAD orthologs in maize and rice were mapped to GABA biosynthesis pathway coinciden- tally (Fig. - These results illustrate that key genes involved in GABA biosynthesis are uniformly stimulated in maize and rice roots under Cd stress.. - To assess the validity of the results from yeast comple- mentation assay, tobacco leaves-based in planta transi- ent analysis was used. - To further explore the common regulatory mecha- nisms in maize and rice under Cd stress, we com- pared Cd-regulated 5166 maize DEGs with their 2567. - counterparts in rice (Additional file 3: Table S3, Additional file 5: Table S5). - As anticipated, the majority of the ortho- logous DEGs in maize and rice showed coordinated expression pattern after Cd treatment. - Notably, 994 out of 1074 maize DEGs with rice counterparts were not stated as being Cd-responsive genes previously (Table 2, Additional file 6: Table S6).. - Among the co-modulated transporters in Cd-treated maize and rice roots, it is noticeable that Cd-responsive HIPPs were categorized into 3 groups of co-upregulated orthologs (Table 2). - a The survival test of yeast strains transformed with ZmGADs on SG-Ura agar medium supplemented with 40 μ M CdCl 2 in the presence of 2% galactose. - The growth of the yeast cells was monitored at OD 600. - RFP-H2A, localized in the nucleus, was used to indicate the nuclei [36]. - It is interesting to note that AtPDR12 and AtPDR8 also are members of the ortho- logs cluster MCL2.. - For in- stance, members of the ZIP family are capable of trans- porting a variety of divalent cations such as Cd, Mn, Fe and Zn ions [49, 50]. - Collectively, the similar expression pattern of these transporter orthologs in Cd-treated maize and rice roots could be as a consequence of the conserved function of orthologous genes. - In the current study, it was interesting to observe that there were several blocks of paralogs co-modulated by Cd stress (Table 1). - Importantly, tandem dupli- cations appear to play an important role in expansion of the GLP family in rice and Arabidopsis [57]. - It is known that GLPs can function as a cofactor for reinforcement of the cell wall through the production of H 2 O 2 due to their SOD activity (e.g. - Additionally, two patatin-like storage proteins are Cd-regulated tandem paralogs on chromosome 2, while the other two are reverse tandem paralogs on chromosome 1, and the latter two were also identified to be Cd-responsive in one previous report [19] (Table 1). - Yet another interesting tandem Cd-responsive paralogs are genes responsible for JA biosynthesis, since both 2 LOXs and 2 AOSs responsible for forming intermediate compounds in JA biosynthesis process are Cd-inducible and clustered on chromosome 1. - Among the 80 universal Cd-responsive DEGs, both ZmOPR2 and ZmOPR5 in cluster MCL166 (Table 1) were also signifi- cantly upregulated in both maize genotypes [19]. - synchronized expression patterns of OPRs and JA-re- sponsive TFs genes have indicated that the JA signaling pathway is one of the crucial elements in the plant re- sponse to Cd stress [24, 71].. - In this context, we proposed that these Cd-responsive paralogs contributed to Cd-tolerance, and their function might be conserved post gene tandem duplication.. - In the present study, SNAC1 and its ortholog in maize were identified to be common stress-responsive as well as Cd-inducible genes (Table 3). - These results collectively suggest that these ortholo- gous genes, simultaneously modulated in maize and rice roots exposed to Cd treatment are also common stress- responsive genes in both species. - However, the majority of the 30 common stress-responsive genes except ClpD and ZmPK1 were not mentioned in previous Cd-stressed maize transcriptomic study (Table 3) [19]. - In the current study, overexpression of maize ZmGAD1 and ZmGAD2 in Cd-sensitive yeast and to- bacco leaves in planta all enhanced Cd tolerance of the host cells (Figs. - These findings implicated that GADs participate in the accumulation of GABA, which contribute to stress acclimation or alleviation.. - Further comparative investiga- tion revealed that ~ 30 maize Cd-responsive genes with rice counterparts were also common stress-responsive genes such as heavy metal-associated domain (HMAD) isopreny- lated protein ZmHIPP27, stress-responsive transcription factor ZmSNAC1, and vp14 (one NCED for ABA biosyn- thesis). - Import- antly, the orthologs of GAD, the key enzyme for GABA synthesis, were concomitantly upregulated in maize and rice roots exposed to Cd treatment. - ZmHIPP27, ZmSNAC1, vp14 and ZmGADs) with rice counterparts were identified to be novel Cd-responsive genes in maize.. - This study extends the understanding of the common mo- lecular mechanisms of plant roots response to Cd and other abiotic stresses, and will be useful for deciphering major candidate genes for improving Cd tolerance in cereal plants.. - Cd-responsive 5166 DEGs in maize roots post Cd treatment. - Cd-responsive 2567 DEGs in rice roots post Cd treatment. - Eight hundred eighty plant orthologous groups composed of Cd-responsive orthologs in maize and rice.. - Global view of Cd-responsive maize orthologs with rice counterparts in metabolic pathways. - None of the members of this funder participated in the study design, data collection and analysis, or preparation of the manuscript.. - The sequence data are available in the NCBI Sequence Read Archive under the accession number of SRP053169 for rice and SRP115510 for maize.. - Cadmium-inducible expression of the ABC-type transporter AtABCC3 increases phytochelatin-mediated cadmium tolerance in Arabidopsis. - Role of the node in controlling traffic of cadmium, zinc, and manganese in rice. - De novo characterization of the Iris lactea var. - Characterization of early transcriptional responses to cadmium in the root and leaf of cd- resistant Salix matsudana Koidz. - Genome-wide analysis of long non-coding RNAs affecting roots development at an early stage in the rice response to cadmium stress.. - Transcriptome analysis of cadmium-treated roots in maize (Zea mays L.).. - Co- expression network analysis of the transcriptomes of rice roots exposed to various cadmium stresses reveals universal cadmium-responsive genes. - A guide to using MapMan to visualize and compare Omics data in plants: a case study in the crop species, maize. - Orthologs of the class A4 heat shock transcription factor HsfA4a confer cadmium tolerance in wheat and rice. - Simultaneous live imaging of the transcription and nuclear position of specific genes. - Transcriptomic profiling of the maize (Zea mays l.) leaf response to abiotic stresses at the seedling stage. - Transport properties of members of the ZIP family in plants and their role in Zn and Mn homeostasis. - Identification and characterization of the zinc-regulated transporters, iron-regulated transporter-like protein (ZIP) gene family in maize. - Importance of lineage-specific expansion of plant tandem duplicates in the adaptive response to environmental stimuli. - Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (cd) hyperaccumulator Noccaea caerulescens. - Functional analysis of the three HMA4 copies of the metal hyperaccumulator Arabidopsis halleri. - Genome-wide characterization and expression analysis of the germinlike protein family in rice and Arabidopsis. - Transgenically expressed rice germin-like protein1 in tobacco causes hyper-accumulation of H2O2 and reinforcement of the cell wall components. - Members of the germin-like protein family in Brassica napus are candidates for the initiation of an oxidative burst that impedes pathogenesis of Sclerotinia sclerotiorum. - C-terminal extension of rice glutamate decarboxylase (OsGAD2) functions as an autoinhibitory domain and overexpression of a truncated mutant results in the accumulation of extremely high levels of GABA in plant cells
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